There is an increasing social concern about the use of recombinant DNA technology. One of the promising application areas of recombinant DNA technology is strain improvement. Starting from the early days of fermentative production processes there has been a demand for the improvement of the productivity of the strains used for production.
Classical strain improvement programs for industrially employed microorganisms are primarily based on random mutagenesis followed by selection. Mutagenesis methods have been described extensively; they include the use of UV light, NTG or EMS as mutagens. These methods have been described extensively for example in “Biotechnology: a comprehensive treatise in 8 vol.”Volume I, Microbial fundamentals, Chapter 5b, Verlag Chemie GmbH, Weinheim, Germany.
Selection methods are generally developed around a suitable assay and are of major importance in the discrimination between wild type and mutant strains.
It has turned out that these classical methods are limited in their potential for improvement. Generally speaking consecutive rounds of strain improvement yield diminishing increases in yield of desired products. This is at least partially due to the random character of the mutagenesis methods employed. Apart from desired mutations these methods also give rise to mutations which are undesirable and which may negatively influence other characteristics of the strains.
In view of these drawbacks it can be understood that the use of recombinant DNA methods was hailed as a considerable improvement. In general, recombinant DNA methods used in strain improvement programs aim at the increased expression of desired gene products.
The gene products may be proteins that are of interest themselves, on the other hand it is also possible that the encoded gene products serve as regulatory proteins in the synthesis of other products.
Strains can be improved by introducing multiple copies of desired protein encoding genes into specific host organisms. However, it is also possible to increase at, expression levels by introducing regulatory genes.
Genes are introduced using vectors that serve as vehicles for introduction of the genes. Such vectors may be plasmids, cosmids or phages. The vector may be capable of expression of the genes in which case the vector generally is self-replicating. The vector may however also only be capable of integration. Another characteristic of the vector is that, when the expression product cannot be selected easily based on altered phenotypic properties, the vector is equipped with a marker that can easily be selected for.
Vectors have not been isolated from all known microorganisms either since no vector could be found in the organism or since available vectors from other organisms could be used with little or no modification. The same applies to selection marker genes.
Widespread use and the subsequent spreading of specific marker genes has recently become debatable. This is especially due to the finding that the use of antibiotics and antibiotic selection markers gives rise to an undesired spread of strains that have become antibiotic resistant. This necessitates the continued development of novel ever more potent antibiotics.
It is therefore not surprising that there is a general tendency in large scale production to use recombinant microorganisms containing no antibiotic resistance genes or more generally as little as possible of foreign DNA.
Ideally the transformed microorganism would contain only the desired gene(s), fragments thereof or modifications in the gene and as little as possible or no further remnants of the DNA used for cloning.